Project Summary This proposal outlines an integrated strategy to gain insights into 3 related processes that determine the levels of integral membrane cell surface. Many cellular activities are exquisitely dependent on the level and repertoire of a variety proteins at the plasma membrane, which reflects a balance of trafficking pathways that control their internalization into endosomes, their transit from endosomes back to the plasma membrane along recycling routes, and their packaging into endosomal intraluminal vesicles prior to delivery and degradation in lysosomes. One of the main determinants for sending membrane proteins for lysosomal degradation is their covalent attachment to ubiquitin, that is subsequently recognized by a series of ubiquitin- sorting receptors. Exactly, how ubiquitinated proteins are physically clustered on endosomal membranes prior to incorporation into intraluminal vesicles has been unclear. However, we have discovered a family of small 4-pass membrane proteins that establish endosomal subdomains that ubiquitinated proteins segregate into. Here we will study the properties of these proteins, the formation of the subdomains, and the types of cargo and sorting events that such subdomains control. Ubiquitin has also been shown to be a signal for internalization, however, the exact sorting receptor(s) that mediate its recognition by many multi-component internalization apparatus. We have discovered that the one family of cargo adaptors for clathrin coated vesicle internalization binds ubiquitin with relatively high affinity. We will now determine whether this family, perhaps in combination with other Ub-binding proteins, serves as a critical component for ubiquitin-dependent internalization of cell surface proteins. One of the critical sorting decisions between degrading a protein or allowing it to remain active takes place in early endosomes, where proteins are either packaged into tubulo-vesicular carriers along a recycling route to the plasma membrane, or packaged into intraluminal vesicles for subsequent delivery and degradation in lysosomes. We recently completed a genetic screen that identified a plethora of new protein machinery required for recycling from early endosomes. This process is under broad metabolic control via the Rag/Gtr GTPases through a process that is independent of TORC1. Here will pursue these observations with biochemical and genetic experiments to connect this metabolic regulation with the trafficking machinery that effects protein movement through the recycling system.